The field of this invention concerns isolated nucleic acid sequences that functions as a transcription enhancer elements for heterologous promoters and methods for using it to identify potential compounds that inhibit expression of native RANTES gene product.
In humans, inflammatory processes are orchestrated in part by a family of soluble mediators called chemokines. One branch of this gene family, the xe2x80x9cCC or xcex1 chemokinesxe2x80x9d, includes RANTES, I-309, the monocyte chemotactic proteins MCP-1, MCP-2, MCP-3 and MCP-4 and the macrophage inflammatory proteins MIP-1xcex1 and MIP-1xcex2. The CC chemokines are pro-inflammatory agents that function as potent and highly selective chemoattractants for specific subsets of hematopoietic cells. MIP-1xcex2 is a chemoattractant for naive helper T cells and MIP-1xcex1 attracts cytotoxic T cells and B lymphocytes, while I-309, MCP-1, MCP-2 and MCP-3 are selective for monocytes. RANTES is chemotactic for monocytes, eosinophils, natural killer cells and the xe2x80x9cmemoryxe2x80x9d population (CD45RO+) of T lymphocytes. RANTES is released from activated platelets and activates basophils to release histamine.
These multiple activities suggest a role for RANTES in both acute and chronic inflammatory processes. Indeed, high concentrations of RANTES have recently been implicated in the control of the human immunodeficiency virus.
RANTES is expressed as an immediate early gene (6-20 hours) by TNFxcex1 stimulated renal tubular epithelium and mesangial cells, TNFxcex1 and IL-1xcex2 activated synovial fibroblasts, and lipopolysaccaride induced monocytes. By contrast, RANTES is expressed in T cells xe2x80x9clatexe2x80x9d (3-5 days) following activation by antigen or mitogen. This xe2x80x9clatexe2x80x9d expression of RANTES by T cells is coincident with the development of T cell effector function, including the expression of perforin and granzymes by cytotoxic T lymphocytes. This pattern of expression is in marked contrast to the immediate early expression (6-20 hours) found for other T cell expressed cytokines, such as IL-2, IL-3, IL-4, IL-5, IL-6, and xcex3IFN or the chemokines I-309, MIP-1xcex1, and MIP-1xcex2.
A general model has been proposed for the pivotal role of RANTES in the induction, amplification and propagation of an inflammatory response. In this scheme, RANTES and other chemokines are induced rapidly within an inflammatory site and bind to the endothelium, where they attract monocytes and T cells by haptotactic mechanisms. In combination with the induced expression of specific integrin and immunoglobulin superfamily members, chemokines lead to mononuclear cell extravasation. The infiltrating cells then follow a haptotactic/chemotactic trail of chemokines into the interstitium. Within days, T lymphocytes, attracted to the inflammatory site, encounter antigen, become activated, differentiate, and strongly upregulate RANTES protein expression. This production of RANTES by T cells then amplifies and propagates the inflammatory response.
Understanding the transcriptional control of an early lymphokine, interleukin-2, has proven a powerful probe into the mechanism of action of the most potent immunosuppressive drugs in clinical use, cyclosporine and FK506. This information has lead to the development of new drugs and the elucidation of pathways involved in the early stages of T cell activation. Understanding the transcriptional control of the late expressed cytokine, RANTES, may similarly provide insight into the molecular pathways involved in later stages of T cell differentiation and lead to the development of novel immunotherapeutics.
The transcriptional machinery controlling RANTES expression differs among the various tissue types capable of expressing this pro-inflammatory cytokine. The large number of potential consensus transcription factor binding sites found within the immediate upstream region of RANTES is unusual and corroborates the multiple points of control of RANTES expression indicated by functional analyses with reporter genes. This complex system for transcriptional control of RANTES expression, with both early and late kinetics in a variety of different tissue types, indicates that diverse activation signals can give rise to a single common pathway of RANTES release, which in turn leads to the attraction of effector cells into an inflammatory site. This single common pathway, therefore, represents an important potential target for inhibition of the inflammatory response.
This invention provides methods for readily identifying compounds that inhibit RANTES expression in T lymphocytes. These compounds can then be used to control undesired inflammatory responses.
For a general introduction to RANTES, see Schall, Cytokine 3:165 (1992), Schall, et al., J. Immunol. 141:1018 (1988) and Nelson, et al., J. Immunol. 151:2601 (1993). Wiedermann, et al., Current Biol. 3:735 (1993) and Pattison, et al., Lancet 343:209 (1994) detail the pivotal role of RANTES in the induction, amplification and propagation of an inflammatory response. And, Nelson, et al., J. Immunol. 151:2601 (1993) describes the genomic organization and transcriptional regulation of the human RANTES gene. Baldwin and Sharp, Proc. Nat. Acad. Sci. 85:723 (1988) and Baeuerle and Henkel, Annu. Rev. Immunol. 12:141 (1994) describe the function and activation of NF-xcexaB. Danoff, et al., J. Immunol. 152:1182 (1994) describes the murine RANTES.
This invention is based on the discovery of nucleic acid sequences present in the human RANTES promoter that mediate upregulation of the RANTES gene late in the T cell developmental pathway.
The present disclosure provides an isolated nucleic acid sequence consisting essentially of the sequence of SEQ ID NO:1, or an isolated nucleic acid sequence consisting essentially of the sequence of SEQ ID NO:2, or an isolated nucleic acid sequence consisting essentially of the sequence of SEQ ID NO:3, or an isolated nucleic acid sequence consisting essentially of the sequence of SEQ ID NO:4, or an isolated nucleic acid sequence consisting essentially of the sequence of SEQ ID NO:5.
In one embodiment, the invention provides an isolated nucleic acid sequence the human RANTES enhancer element R(A) (SEQ ID NO:2), said sequence defined by its ability to inducibly express a gene when positioned upstream of the CAAT and TATA boxes of a minimal promoter with the promoter operably linked to the gene to form an expression cassette where the expression cassette is transfected into an activated peripheral blood lymphocyte cell which has been contacted with an amount of anti-CD3 antibody sufficient to induce the expression of the gene with the proviso that the nucleic acid sequence is not operably linked to the RANTES gene product nor to the native RANTES promoter sequences. This R(A) element can be operably linked to a heterologous gene. It can be positioned downstream of a
CAAT box and upstream of a TATA box and further separated from said TATA box by a functional NFxcexaB binding site. The NFxcexaB binding site can be the native RANTES NFxcexaB site which has been rendered non-functional.
The R(A) element can also be combined with additional transcriptional elements to form an artificial RANTES promoter. Three elements that can be operably linked to the R(A) element are: (1) the R(C) binding site which is SEQ ID NO:3 corresponding to xe2x88x92182 to xe2x88x92169 of the human Rantes promoter; (2) the Region xe2x80x9cCxe2x80x9d sequence corresponding to xe2x88x92195 to xe2x88x92144 of the human Rantes promoter which is SEQ ID NO:4; or (3) the Region E sequence corresponding to xe2x88x92115 to xe2x88x9291 of the human Rantes promoter which is SEQ ID NO:5.
This R(A) element as well as the above-identified transcriptional regions can be recombined with additional nucleic acid sequence to form a vector. The R(A) element can be operably linked to a DNA sequence which encodes a heterologous protein. The heterologous protein can be selected from the group consisting of: hormones, viral capsid proteins, bacterial enzymes and mammalian enzymes. The vector containing the R(A) element recombined with additional nucleic acid sequence can be transfected into a host cell. Said host cell can be a mammalian cell competent to express the binding elements needed to induce expression of the heterologous protein. Said cell can be activated peripheral blood lymphocytes or T cell tumor line Hut78.
Methods for inducing the expression of heterologous proteins and for detecting inhibitors of RANTES production are also provided by this invention.
This invention provides a method for inducing the expression of a heterologous protein in a host cell having nucleic acid sequence encoding the heterologous protein wherein said nucleic acid sequence is operably linked to a promoter comprising the R(A) enhancer element derived from the nucleic acid sequence encoding the human RANTES protein and further defined by (a) the ability to inducibly express a gene when operably linked to a gene forming an expression cassette and (b) the ability of the expression cassette when transfected into a peripheral blood lymphocyte in the presence of anti-CD3 antibody to induce the expression of the gene, with the proviso that the nucleic acid is not operably linked to the RANTES gene product nor to the native NFxcexaB binding element of the RANTES gene, said method comprises: (i) transfecting the host cell with the expression cassette having nucleic acid sequence encoding the heterologous protein; and (ii) inducing the expression of the heterologous protein.
Other methods of inducing the expression of heterologous proteins include those that have SEQ ID NO:2 for the R(A) element. They also include those where said host cell is selected from the group consisting of activated peripheral blood lymphocytes and T cell tumor line Hut78, or said host cell is transfected with a plasmid. Additionally, said heterologous protein can be selected from the group consisting of: hormones, viral capsid proteins, bacterial enzymes and mammalian enzymes. These methods further include those where said nucleic acid sequence is transfected into a human host cell or where the host cell is induced to express a heterologous gene by contacting the cell with an appropriate activator in an amount sufficient to induce expression of the gene.
To obtain optimal control of expression of the RANTES R(A) element, one can add any of the above transcriptional elements.
This invention also provides a method for detecting inhibitors of RANTES production by inducing the expression of a heterologous protein in a host cell having the nucleic acid sequence encoding the heterologous protein wherein said nucleic acid sequence is operably linked to a promoter comprising the R(A) element from the promoter of the gene encoding the human RANTES protein, wherein the promoter does not include the other native cis binding sites of the RANTES gene and is further defined by (a) the ability to inducibly express a gene when operably linked to a gene forming an expression cassette and (b) the ability of the expression cassette when transfected into a peripheral blood lymphocyte in the presence of anti-CD3 antibody to induce the expression of the gene, said method comprises: (i) transfecting the host cell with the expression cassette having nucleic acid sequence encoding the heterologous protein; (ii) choosing a composition of matter that potentially has inhibitory effect on RANTES expression; (iii) inducing the expression of the heterologous protein in the presence of said potential inhibitor; and (iv) assaying for decreases in the level of expression. The above described transcriptional elements, i.e., the C Region, the C binding site and the E Region may all be combined with the R(A) element to ensure that the inhibitor has the desired specificity.